Medical Microbiology

Microbial Pathogenicity

Lecture #14 Bio3124

Pathogens as Parasites •

pathogens are parasites

– organisms that live on or within a host organism, metabolically dependent on the host – Parasitism: • Ectoparasite: parasite lives on the host • Endoparasite: parasite lives in the host

Parasitism and disease

• Infection – growth and multiplication of parasite on or within host • Infectious disease – disease resulting from infection • Pathogen: any parasitic organism that causes infectious disease – primary (frank) pathogen – causes disease by direct interaction with healthy host – opportunistic pathogen – part of normal flora, causes disease when gains access to other tissue sites or when host is immunocompromised • Pathogenicity – ability of a parasite to cause disease

Host-parasite relationship and disease outcome Disease state depends on :

– number of organisms present – degree of virulence of pathogen – virulence factors • e.g., capsules, pili, toxins – host’s defenses or degree of resistance

Virulence:

degree/intensity of pathogenicity • determined by, – Invasiveness: ability to spread to adjacent tissues – Infectivity: ability to establish focal point of infection – pathogenic potential: degree to which pathogen can cause damage to host • Toxigenicity: ability to produce toxins • Immunopathogenicity: ability to trigger exaggerated immune responses

Measuring virulence

• lethal dose 50 (LD 50 ) – number of pathogens that will kill 50% of an experimental group of hosts in a specified time • Infectious dose 50 (ID 50 ) – number of pathogens that will infect 50% of an experimental group of hosts in a specified time

Infection Cycle

• Mode of entry depends on pathogen • Mucosal surfaces, wounds, insect bites •

Infection cycle

Route a pathogen takes to spread • Spread via direct contact • Indirect contact – Contact with fomites – Horizontal transmission via vectors • Mosquitoes—Yellow fever, malaria • Reservoir for disease organism – May not show disease symptoms

Virulence Factors

• Virulence genes

– Help pathogen to invade host • Toxins, attachment proteins, capsules

• Pathogenicity islands

– Section of genome • Contain multiple virulence genes – Often encode related functions » protein secretion system, toxin production – Horizontally transmitted • Often flanked by tRNA genes; phage or plasmid genes • Often have GC content different from rest of genome

Virulence Factors

• Several factors contribute – Protein secretory systems • Examples:Type II, type III and type IV – Adhesins: host attachement & colonization – Toxins • Exotoxins – Membrane active toxins – Protein synthesis inhibitors – Cell signaling inhibitors – Superantigens – proteases • Endotoxins – Immune avoidance factors

Role of protein secretory pathways in virulence

• PS Type II (retractable) – Subunits in inner, outer and periplasmic space – G subunit polymerize/depolymerize – Extends/retracts past outer membrane through complex D – like a piston pushes out the secreted proteins to periplasmic space – Ex. Cholera toxin • PS Type II mechanism resemble pili type IV used for twitching motility

Type III protein secretory system

• many G- bacteria, live in close association with their hosts • secrete regulatory proteins via injectisome directly into host cells – to modulate host cell activities – evolutionary resemblance to flagellum • increase virulence potential – Avoids receptor use – Avoids dilution of secreted proteins outside pathogen

Ken Miller talks about PSIII and flagellum

Salmonella SPI-1 and SPI-2 are type III secretory systems • 12 pathogenicity islands in

S. typhi

• SPI-1 , a type III secretory system • Injects 13 different toxins (effector proteins) • Subvert signaling, remodel cytoskeleton • Induce membrane ruffles, take

S.typhi

• SPI-2: alter vesicle trafficking – Prevent phogosome-lysosome fusion – Pathogen avoids innate immunity

Injectisome: a type III secretory virulence factor

Toxin secretion by type IV secretory system

• • Resemble conjugation apparatus of gram negative bacteria

Bordetella pertussis

toxin secreted through general SecA pathway to periplasm • Type IV collects toxin in periplamic space • Exports across outer membrane

General SecA dependent secretory system

Adhesins: Microbial Attachment

• Human body expels invaders – Mucosa, dead skin constantly expelled – Liquid expelled from bladder – Coughing, cilia in lungs – Expulsion of intestinal contents • Adhesins: surface proteins, glycolipids, glycoproteins – assist in attachment and colonization of host tissues • Pili (fimbriae) • Hollow fibrils with tips to bind host cells

Adhesins: Pili type I

• e.g. Pyelonephritis pili of uropathogenic

E.coli

• attachment to P-blood group antigen • upper uninary tract infection • • Pili assemble on outer membrane First, general SecA dependent secretion to periplasm • • PapG,E,F & major subunit Pilin A PapD chaperon sorting/delivery to PapC • Secretion and pilus formation • PapG recognizes the digalactoside on P-blood group antigen of host kidney cells

Adhesins: Pili type IV

• Found on

P. aeruginosa

,

V. cholera,

pathogenic

E. coli & N. meningitidis

• Mediates attachment and twitching motility • Resemble type II secretory system • Pil A is major structural pilin • PilC,Y1 tip attachment proteins • Assembly: PilA preprotein signal sequence removed by PilD • PilQ mediates export across outer membrane • PilF/T mediates energy dependent assembly/disassembly of pilus

Type IV pili: bacterial attachment and motility

Exotoxins

• soluble, heat-labile, proteins • usually released into the surroundings as bacterial pathogen grows • most exotoxin producers are gram positive • often travel from site of infection to other tissues or cells where they exert their effects

More About Exotoxins

• Some toxin genes born on plasmids or prophages • the most lethal substances known • highly immunogenic • can stimulate production of neutralizing antibodies (

antitoxins

) • can be chemically inactivated to form immunogenic

toxoids

– e.g., tetanus toxoid

Membrane-disrupting exotoxins

• • Alpha toxin of

S. aureus

Forms 7-membered oligomeric beta-barrel Cause cytoplasmic leakage Phospholipase of

Clostridium perfringens

• removes charged head group of phospholipids in host-cell plasma membranes – membrane destabilized, cell lyses and dies – Also called α-toxin or lecithinase

AB type Exotoxins

Composed of two subunits

• “ A ” subunit – responsible for toxic effect – ADP-ribosyltion of target proteins eg. diphtheria toxin – Cleave 28S rRNA, eg. Shiga toxin • “B” subunit – binds to target cell, delivers A subunit Diphtheria exotoxin • B subunit mediates receptor binding • Endocytosis and fusion membrane vesicles eg. ER or endosomes • B recycles back to membrane • “A” escapes and enters cytoplasm • In the cytoplasm A catalyses ADP ribosylation of EF2, halts translation • Cell death ensues

Diphtheria toxin targets EF2 disrupts translation

Anthrax toxin: a deadly protease • Anthrax toxin composed of,

– Protective antigen

(B subunit)

: delivers EF and LF (A subunits) – Edema factor raises cAMP levels • Causes fluid secretion, tissue swelling – Lethal factor cleaves protein kinases • Blocks immune system from attacking

Bacillus anthracis

Animation:

anthrax toxin mode of action

Superantigens

• Are bacterial and viral proteins that can activate T-cells • in the absence of a real bacterial antigen mediate the binding of MHC-II and T-cell receptors (almost 30% of T cell population) • eg.

Staphylococcal

enterotoxin B (SEB) • Massive activation results in producing lots of cytokines • Results in tissue damage and shock and multi-organ failure

Animation: Superantigens

Endotoxins

• lipopolysaccharide in gram-negative cell wall can be toxic to specific hosts – called endotoxin because it is bound to bacterium and released when organism lyses and some is also released during multiplication – toxic component is the lipid portion, lipid A • heat stable • toxic (nanogram amounts) • weakly immunogenic • generally similar, despite source

Immune avoidance mechanisms • Once inside host cell, how to avoid death?

– Cell ingests pathogens in phagosome • Some pathogens use hemolysin to break out –

Shigella dysenteriae

,

Listeria monocytogenes

– Phagosome fuses with acidic lysosome • Some pathogens secrete proteins to prevent fusion –

Salmonella

,

Chlamydia

,

Mycobacterium

,

Legionella

• Some pathogens mature in acidic environment –

Coxiella burnetii

—Q fever

Surviving within the Host

Survival inside phagocytic cells

• escape from phagosome before fusion with lysosome – microbes use actin-based motility to move within and spread between mammalian host cells

Burkholderia pseudomallei

forming actin tails and protrude through membrane and extend infection to nearby cells

Surviving within the Host

• Outside host cell, how to avoid death?

– Complement, antibodies bind pathogen • Some pathogens secrete thick capsule –

Streptococcus pneumoniae

,

Neisseria meningitidis

• Some pathogens make proteins to bind antibodies –

Staphylococcus aureus

cell wall Protein A » Binds Fc fragment » Antibodies attach “upside down” » Prevents opsonization • Some pathogens cause apoptosis of phagocytes